Application of total flavonoid extract of Ziziphora clinopodioides Lam. in preparing medicine for treating cerebral ischemia-reperfusion injury

Abstract

Application of total flavonoid extract of Ziziphora clinopodioides Lam. in the preparation of drugs for treating cerebral ischemia-reperfusion injury. The present invention belongs to the field of cerebrovascular diseases, and particularly relates to the application of total flavonoid extract of Ziziphora clinopodioides Lam. (ZCF) in the preparation of drugs for treating cerebral ischemia-reperfusion injury. The present invention has studied the effects of different doses of ZCF against cerebral ischemia-reperfusion injury. The research shows that ZCF has a significant therapeutic effect on Sprague-Dawley rats with a middle cerebral artery occlusion-reperfusion (MCAO / R) model prepared by the suture method, significantly reduces the cerebral infarction area (Figure 1), reduces the blood-brain barrier permeability, and can effectively improve cognitive and motor dysfunction. The present invention also provides a drug that effectively resists cerebral ischemia-reperfusion injury, and can provide a reliable experimental basis and theoretical basis for the development of drugs for treating cerebral ischemia-reperfusion injury using Ziziphora clinopodioides Lam.

Description

Technical Field

[0001] This application relates to the technical field of cerebrovascular diseases, and particularly to the application of the total flavonoid extract of Ziziphora clinopodioides Lam. in the preparation of a drug for treating cerebral ischemia-reperfusion injury. Background Art

[0002] Stroke, also known as apoplexy, is an acute cerebrovascular disease. According to different pathogenesis, stroke is divided into ischemic stroke and hemorrhagic stroke. The incidence of ischemic stroke is higher than that of hemorrhagic stroke, accounting for 60% - 70% of the total number of strokes, and it is the most common type of stroke. At present, there are few treatment strategies for ischemic stroke. Revascularization to restore blood supply is the main measure for treating ischemic stroke. However, both the early stage of cerebral ischemia and the process of blood reperfusion after treatment have a great risk of causing secondary reperfusion injury, which is called cerebral ischemia-reperfusion injury.

[0003] The mechanisms involved in cerebral ischemia-reperfusion injury include free radical injury, neuroinflammation, excitotoxicity, and neuronal apoptosis, etc. Some studies have shown that cerebral ischemia-reperfusion injury can increase the area of cerebral infarction, damage the blood-brain barrier, aggravate brain tissue damage, and cause cognitive and motor dysfunction through the above-mentioned series of pathological pathways. Cerebral ischemia-reperfusion injury is one of the important factors leading to severe disability and death of patients. The treatment of cerebral ischemia-reperfusion injury has always been a key task in the treatment of ischemic stroke. Clinically, there is still a lack of effective drugs for the treatment of reperfusion injury of ischemic stroke. Finding drugs that may intervene in the pathophysiological mechanism of ischemic stroke is a reliable way to improve ischemic brain injury.

[0004] As a traditional Chinese medicine, Ziziphora clinopodioides Lam. is mainly distributed in Xinjiang and has a long history of being widely used in Xinjiang folk for the treatment of cardiovascular diseases. According to the domestic scholars He Jiang et al., after extracting Ziziphora clinopodioides Lam. and using HPLC-MS / MS to identify the components of the extract, it was shown that 12 flavonoid compounds had relatively high peak areas in the extract of Ziziphora clinopodioides Lam., and they could be used as the main flavonoid components of Ziziphora clinopodioides Lam., namely: (a) kaempferol-7-O-rutinoside; (b) kaempferol-3-O-rutinoside; (c) rutin; (d) apigenin-7-O-rutinoside; (e) 3'-hydroxyacacetin-7-O-rutinoside; (f) acacetin-4'-O-rutinoside; (g) pinocembrin-7-O-rutinoside; (h) chrysin-7-O-rutinoside; (i) buddleoside; (j) 5,7,3'-trihydroxy-6,4',5'-trimethoxyflavone; (k) 5,4'-dihydroxy-6-methoxy-7,8-methylenedioxyflavone; (l) 5,7-dihydroxy-6-methoxyflavone.

[0005] In recent years, studies have found that the main flavonoid compounds in Ziziphora suaveolens (including quercetin, baicalein, etc.) have significant pharmacological effects such as anti-inflammatory and antioxidant effects. For example, quercetin has been proven to have a neuroprotective effect on glutamate-induced hippocampal-derived (HT22) cells, can significantly improve the neurobehavioral deficits caused by cerebral ischemia, and reduce the volume of cerebral infarction. Baicalein can reduce the cell damage induced by oxygen-glucose deprivation / reoxygenation (OGD / R), and inhibit ferroptosis through multiple pathways such as reducing iron content, inhibiting lipid peroxidation reaction, increasing endogenous antioxidant activity, and regulating the expression of multiple ferroptosis-related proteins to alleviate cerebral ischemia-reperfusion injury, etc. However, there is currently no report on the application of the total flavonoid extract of Ziziphora suaveolens (ZCF) in anti-cerebral ischemia-reperfusion injury. Summary of the Invention

[0006] Based on this, the present invention provides a new use of ZCF, which relates to the application of ZCF in the preparation of drugs for preventing, alleviating or treating cerebral ischemia-reperfusion injury.

[0007] The specific technical solutions are as follows:

[0008] In the first aspect of the present application, there is provided the application of ZCF in the preparation of drugs for anti-cerebral ischemia-reperfusion injury. Secondly:

[0009] In one embodiment, the anti-cerebral ischemia-reperfusion injury includes improving cognitive and motor dysfunction.

[0010] In one embodiment, the anti-cerebral ischemia-reperfusion injury includes reducing the area of cerebral infarction.

[0011] In one embodiment, the anti-cerebral ischemia-reperfusion injury includes reducing the permeability of the blood-brain barrier.

[0012] In one embodiment, the ZCF is a mixture of alcohol-extracted flavonoid components.

[0013] In one embodiment, the dosage form of the drug for anti-cerebral ischemia-reperfusion injury is an oral preparation, which is mainly composed of ZCF and a solvent.

[0014] The present invention has studied the effect of different doses of ZCF on anti-cerebral ischemia-reperfusion injury. The research shows that ZCF has a significant therapeutic effect on SD rats with a middle cerebral artery occlusion-reperfusion (MCAO / R) model prepared by the suture method, significantly reduces the area of cerebral infarction ( Figure 1 ), reduces the permeability of the blood-brain barrier, and can effectively improve cognitive and motor dysfunction. The present invention also provides a drug that effectively resists cerebral ischemia-reperfusion injury, which can provide a reliable experimental basis and theoretical basis for the development of drugs using ZCF for anti-cerebral ischemia-reperfusion injury.

[0015] Compared with the prior art, the present invention first applies ZCF to anti-cerebral ischemia-reperfusion injury. The researchers of this application used a rat middle cerebral artery occlusion / reperfusion (MACO / R) model to evaluate the effect of ZCF on anti-cerebral ischemia-reperfusion injury. The main detection indexes include behavioral evaluation, determination of cerebral infarction area and detection of blood-brain barrier permeability. The research results prove that ZCF has a significant repair effect on cerebral ischemia-reperfusion injury, can significantly reduce the cerebral infarction area, reduce the blood-brain barrier permeability, improve cognitive and motor dysfunction, and can be applied to the preparation of drugs for anti-cerebral ischemia-reperfusion injury. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Structural formulas of 12 main flavonoid components in ZCF.

[0017] Figure 2 It is a graph of the experimental results of the behavioral evaluation of rats with ZCF against cerebral ischemia-reperfusion injury involved in the present invention. Among them, A is the behavioral trajectory graph of the open field test, B is the statistical graph of the slow movement time in the open field test, C is the statistical graph of the scores of the balance beam test, D is the statistical graph of the sticker removal time of the sticker removal test, and E and F are the statistical graphs of the complete turning time (T-turn) and landing time (TD) of the pole climbing test respectively.

[0018] Figure 3 It is a graph of the experimental results of the TTC staining of the cerebral infarction area of rats with ZCF against cerebral ischemia-reperfusion injury involved in the present invention.

[0019] Figure 4 It is a graph of the experimental results of the Evans blue staining of rats with ZCF against cerebral ischemia-reperfusion injury involved in the present invention.

[0020] DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] The following further details the application of ZCF of this application in the preparation of drugs for anti-cerebral ischemia-reperfusion injury in combination with specific embodiments. This application can be implemented in many different forms and is not limited to the embodiments described herein. The purpose of providing these embodiments is to help understand the disclosure of this application more thoroughly and comprehensively.

[0022] Example 1

[0023] This example relates to the preparation of the total flavonoid extract of Ziziphora clinopodioides Lam. (ZCF), specifically: an appropriate amount of Ziziphora clinopodioides Lam. herbs, extracted twice with 20 times the amount of 70% ethanol by heating for 1.5 hours each time, the filtrate was concentrated and dried under reduced pressure.

[0024] The structural formulas of 12 main flavonoid components contained in the total flavonoid extract of Ziziphora clinopodioides Lam. (ZCF) are as Figure 1 shown.

[0025] Example 2

[0026] 1. Animal feeding

[0027] SD rats were purchased from the Experimental Animal Center of Hangzhou Medical College and raised in an SPF - level laboratory. During the feeding period, water and food were freely supplied, and the light - dark cycle was 12 h.

[0028] 2. Establishment of MCAO model

[0029] The suture occlusion method was used to prepare the MCAO model in rats. A suture was prepared by selecting a No. 2.5 nylon fishing line with a diameter of 0.260 mm. The head end was heated with an alcohol lamp to form a hemispherical shape and coated with silicone grease. For 250 - 300 g rats, a transient middle cerebral artery occlusion / reperfusion (MCAO / R) model in rats was established as follows Figure 3 As shown below, the specific operation was as follows: 250 - 300 g male SD rats were anesthetized with 4% chloral hydrate (0.85 ml / 100 g). Under anesthesia, the rats were fixed in the supine position. The skin was incised in the middle of the neck, and the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were bluntly dissected, and careful dissection was carried out to avoid damaging the vagus nerve. A fine nylon suture was slowly inserted from the ECA and advanced forward to 18 mm of the ICA until slight resistance was encountered, causing focal cerebral ischemia in the rats. The rats were maintained on a small animal heating blanket. After 90 min, the suture was withdrawn to achieve reperfusion of the middle cerebral artery (MCA). The sham - operation group only exposed the common carotid artery as a control. After the animals recovered from the modeling and showed obvious hemiplegia symptoms, such as body tilt and crawling rotation, it was considered that the modeling was successful, or neurobehavioral examination was performed by the Longa scoring method.

[0030] 3. Grouping and drug administration

[0031] Sixty male SD rats were purchased and adaptively fed for 1 week. Except for the sham - operation group (Sham group) where only the common carotid artery, internal carotid artery, and external carotid artery were exposed and separated during the operation without embolization treatment, the remaining rats were all constructed with the MCAO / R model. The modeled rats were randomly divided into 5 groups, namely the model group (Vehicle group), ZCF - 75 mg / kg group, ZCF - 150 mg / kg group, ZCF - 300 mg / kg group, and the positive control group of butylphthalide, with 10 rats in each group. Gastric gavage administration started on the second day after modeling. Among them, the ZCF - administered groups were given different doses of ZCF by gastric gavage, the positive control group was given butylphthalide by gastric gavage, and the normal control group and the model control group were given the corresponding solvents by gastric gavage for 1 week continuously. During this period, water and food were freely supplied.

[0032] 4. Research methods and related detection indexes

[0033] (1) Behavioral experiments were used to evaluate the learning, memory, and cognitive function levels of rats after MCAO / R

[0034] ① Open field test was used to detect the cognitive and motor abilities of rats

[0035] Rats in each group were placed in the experimental box in advance to adapt to the experimental environment. Then the rats were placed in the center of the test area, and their activity trajectories in the test area were recorded. The test time was 5 min. The slow activity time of the rats was recorded using connection analysis software program, and the number of times and duration of the rats entering the middle area were statistically analyzed, and the trajectory map was drawn.

[0036] ② Sticker test was used to detect the motor ability of rats

[0037] Two square stickers with a side length of 10 mm were stuck on the palm surfaces of the two front limbs of the rats, and the viscosity was such that normal rats could bite off the stickers within 10 s. The rats were placed in a test bucket, and the total time for the rats to instinctively remove the stickers on both sides was observed and recorded. If the stickers were not removed within more than 120 seconds, it was recorded as ......

[0038] ③ Balance beam test was used to detect the motor coordination ability of rats

[0039] A balance beam with a length of 80 cm and a width of 2.5 cm was placed horizontally at a height of 10 m from the ground. According to Feeney's scoring criteria: 0 points means passing through the balance beam without falling; 1 point means passing through the balance beam with a fall chance of less than 50%; 2 points means passing through the balance beam with a fall chance of more than 50%; 3 points means being able to pass through the balance beam, but the affected paralyzed hind limb cannot help move forward; 4 points means not being able to pass through the balance beam, but can sit on it; 5 points means the rat will fall when placed on the balance beam. The scores of rats in each group were statistically analyzed.

[0040] ④ Pole climbing test was used to detect the cognitive and motor abilities of rats

[0041] The experimental device for the pole climbing test was established. The balance pole was obliquely placed between the top of the iron frame and the base, and the rat's head was placed upwards at the top of the pole. The rat naturally oriented downwards and descended along the length of the pole, and the time for the rat to completely turn downwards (T - turn) and the time required to descend to the bottom of the pole (TD) were recorded.

[0042] From Figure 2 It can be seen from each figure that compared with the sham - operation group, the cognitive and motor functions of the model group were significantly decreased. After administration of different doses of ZCF, the cognitive and motor functions of the rats were significantly improved. It shows that ZCF can improve the cognitive and motor functions of rats with cerebral ischemia - reperfusion injury.

[0043] (2) TTC staining method was used to detect the level of cerebral infarction

[0044] On the 7th day after MCAO / R surgery in rats, each group of rats was sacrificed by overdose anesthesia with chloral hydrate and perfused with normal saline through the heart. Brain slices were cut at a position 12 mm in front of the interaural line of the brain (the sensory and motor areas of the frontal cortex) to 510 mm (the middle of the hippocampus), with a thickness of about 2 mm. Then the brain slices were placed in 4% 2,3,5-triphenyltetrazolium chloride (TTC) phosphate buffer solution and incubated at 37°C in the dark for 30 min. After that, they were taken out and fixed in 10% paraformaldehyde solution and photographed.

[0045] From Figure 3 As can be seen from each figure, compared with the sham operation group, obvious white infarcted areas appeared in the brain tissues of the model group rats after TTC staining. After administration of different doses of ZCF, the white areas were significantly reduced. This indicates that ZCF reduces the cerebral infarction area in rats with cerebral ischemia-reperfusion injury.

[0046] (3) Observation of blood-brain barrier permeability changes by Evans blue (EB) staining

[0047] Rats were injected with Evans Blue Stain (0.5%) at a dose of 5 ml / kg via the caudal vein. Within several seconds to 1 minute, the rats' eyes and skin turned blue. The rats were sacrificed 0.5 - 1 h later, and brain tissue sections were taken for observation.

[0048] From Figure 4 As can be seen from each figure, compared with the sham operation group, obvious Evans blue leakage appeared in the brains of the model group rats. After administration of different doses of ZCF, the Evans blue leakage decreased, indicating that ZCF reduces the blood-brain barrier permeability in rats.

[0049] In summary, under the conditions of this experiment, ZCF has a significant repair effect on MCAO / R injury in rats, can significantly reduce the cerebral infarction area, decrease the blood-brain barrier permeability, improve cognitive and motor dysfunction, and can be developed as a drug for anti-cerebral ischemia-reperfusion injury.

[0050] The technical features of each of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as within the scope described in this specification. The above-described embodiments only express several implementation manners of the present application, which are convenient for understanding the technical solutions of the present application specifically and in detail, but should not be construed as a limitation on the scope of patent protection of the application. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present application, several modifications and improvements can still be made, and these all belong to the protection scope of the present application. It should be understood that the technical solutions obtained by those of ordinary skill in the art through logical analysis, reasoning or limited experiments based on the technical solutions provided in the present application are all within the protection scope of the appended claims of the present application.

[0051] Therefore, the scope of protection of this application shall be subject to the content of the appended claims, and the description and drawings may be used to interpret the content of the claims.

Claims

1. Use of the total flavonoid extract of Ziziphora clinopodioides Lam. in the preparation of a drug for treating cerebral ischemia-reperfusion injury, characterized in that, The preparation method of the total flavonoid extract of Ziziphora clinopodioides Lam. is as follows: The medicinal materials of Ziziphora clinopodioides Lam. are extracted twice with 20 times the amount of 70% ethanol by heating for 1.5 hours each time, the filtrate is concentrated, and then dried under reduced pressure.

2. The application according to claim 1, characterized in that The dosage form of the drug for treating cerebral ischemia-reperfusion injury is an oral preparation.

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